Haplotype characterization of phosphorus homeostasis gene, SPX-MFS3 under combination of low nitrogen and phosphorus conditions in indica rice at seedling stage.

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Learn more: PMC Disclaimer | PMC Copyright Notice 3 Biotech . 2025 May 26;15(6):188. doi: 10.1007/s13205-025-04350-1 Haplotype characterization of phosphorus homeostasis gene, SPX-MFS3 under combination of low nitrogen and phosphorus conditions in indica rice at seedling stage Baishnab Charan Muduli 1, 2 , Sabarinathan Selvaraj 1 , Shivraj Sahu 1 , Subhadarshani Dhall 3 , Nibedita Swain 1 , Parameswaran Chidambaranathan 1, ✉ , Jitendriya Meher 1 , Chinmay Pradhan 2 , Dibyendu Chatterjee 1 , Sangita Mohanty 1 , Sanghamitra Samantaray 1 Author information Article notes Copyright and License information 1 ICAR- National Rice Research Institute, Cuttack, Odisha 753006 India 2 Department of Botany, Utkal University, Vani Vihar, Bhubaneswar, Odisha 751004 India 3 Centre for Biotechnology, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha 751004 India ✉ Corresponding author. Received 2024 Oct 4; Accepted 2025 May 5; Issue date 2025 Jun. © King Abdulaziz City for Science and Technology 2025. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. PMC Copyright notice PMCID: PMC12106286  PMID: 40438160 Abstract Plants establish a complex signalling network to acquire nitrogen (NO 2 ⁻/NH 4 ⁺) and phosphate (Pi) from the soil. In rice, OsSPX-MFS3 (LOC_Os06g0129400) has been characterized as a vacuolar low-affinity Pi transporter gene, which plays a crucial role under phosphorus-deficient conditions. This study aimed to identify genetic variants and favourable haplotypes of the OsSPX-MFS3 gene (Chr06: 1554861-1561163) associated with seedling traits under combined low N and low P conditions. A total of 99 genetic variants of OsSPX-MFS3 were identified across 223 indica rice accessions, and their association with eight seedling traits was analysed under low N and low P conditions at 28 and 42 days after sowing (DAS) in Kharif and Rabi seasons. Among the eight major OsSPX-MFS3 haplotypes, significant differences were observed for three traits (shoot length (0.01*), shoot area (0.05*), and root area (0.02*)) in kharif and six traits (Root length (0.03*), shoot area (0.003**), root area (0.02*), shoot dry weight (0.002**), root dry weight (0.01*), and SPAD (0.005**,0.03*) in kharif and rabi seasons. Further, Hap_5 was identified as a favourable haplotype at the seedling stage under combined N and P deficiency. Promoter cis-regulatory analysis of Hap_5 identified unique cis-regulatory elements (ERE, MYC, O 2 -site, MYM, and ABRE) relative to other haplotypes. Besides, three tolerant indica accessions (IRGC 131940, IRGC 126966, and IRGC 122165) were identified for better shoot and root growth under nutrient-deficient conditions. These findings highlight the potential application of OsSPX-MFS3 haplotypes in haplotype-based breeding programs aimed at developing rice cultivars with improved tolerance to low nitrogen and phosphorus conditions. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-025-04350-1. Keywords: Haplotype analysis, SPX-Major Facility Superfamily3 , Low Nitrogen, Low Phosphorus, Seedling stage, Marker trait association Introduction Rice ( Oryza sativa L.) is a staple cereal crop that provides essential nutrition to nearly half of the global population (Shin et al. 2020 ). Ensuring food security in the coming decades remains a critical challenge, particularly with the increasing global population (Godfray et al. 2010 ). Nutrient availability plays a fundamental role in shaping plant growth, influencing gene expression, and ensuring optimal agricultural productivity (Xuan et al. 2017 ). Among essential nutrients, nitrogen (N) and phosphorus (P) are key macronutrients required for plant growth and development. Nitrogen is a crucial element involved in numerous physiological and biochemical processes, as it forms the structural basis of nucleotides, amino acids, and chlorophyll (Tariq et al. 2023 ). It is particularly important during the early vegetative stage of rice growth (Wang et al. 2023 ). Rice plants absorb N primarily in the form of nitrate (NO₃⁻) and ammonium (NH₄⁺) from the soil (Wang et al. 2012 ; Fan et al. 2016 ). The efficiency of nitrogen uptake from the soil directly influences the agronomic traits such as plant height, leaf area, canopy area, root length, and root area (Chakwizira et al. 2023 ; Keerthi et al. 2023 ). Nitrogen use efficiency (NUE) in rice is a complex trait governed by multiple genes and quantitative trait loci (QTLs) that regulate interactions between genetic and environmental factors (Sandhu et al. 2021 ; Sharma et al. 2021 ; Phan et al. 2023 ). Phosphorus is another vital macronutrient required for various biological functions, including energy metabolism, redox reactions, photosynthesis, and phosphorylation/dephosphorylation processes (Liu et al. 2015 ; Kleinert et al. 2017 ). In plants, P is absorbed from the soil in the form of inorganic phosphate (Pi), but its availability is highly dependent on soil pH and water conditions (Ceasar 2020 ). Phosphorus plays a critical role in nucleic acid structure, high-energy compound synthesis, enzyme regulation, and membrane stability. Phosphorus deficiency significantly affects early seedling growth, influencing traits such as shoot length, root length, and chlorophyll content (Anandan et al. 2021; Sandhu et al. 2021 ). Recent studies have identified vacuolar Pi transporters (VPTs), also known as Pi transporter type 5 ( PHT5 ) are involved in phosphorus homeostasis. These transporters contain both SYG1/PHO81/XPR1 ( SPX ) and the major facilitator superfamily ( MFS ) domains, enabling Pi sequestration into vacuoles in Arabidopsis (Liu et al. 2015 ; Liu et al. 2016 ) and rice (Secco et al. 2012 ).The SPX domain, named after Suppressor of Yeast gpa1 (SYG1), Phosphatase 81 (PHO81), and Xenotropic and Polytropic Retrovirus receptor 1 (XPR1), is widely present in phosphate transporters and signaling proteins in plants (Secco et al. 2012 ). In rice, SPX-domain proteins are categorized into four subfamilies: SPX, SPX-RING, SPX-MFS, and SPX-EXS (Chen et al. 2023 ). Among the SPX-MFS family members, OsSPX-MFS3 has been identified as a key regulator of Pi transport and homeostasis. It functions as a vacuolar low-affinity Pi transporter, mediating Pi efflux from the vacuole to the cytosol in a proton-coupled manner (Wang et al. 2015 ; Guo et al. 2023a ). The expression of OsSPX-MFS3 is downregulated under Pi deficiency, and mutations in this gene have been shown to reduce Pi remobilization in rice (Du et al. 2017 ). Furthermore, the SPX protein family is linked to nitrogen regulation, as Nitrogen Limitation Adaptation ( NLA ) gene, a member of the SPX family, plays a role in nitrate-dependent Pi homeostasis in Arabidopsis (Ueda et al. 2020 ). A haplotype is a combination of alleles for different polymorphisms (such as SNPs, insertions/deletions and other markers or variants) on the same chromosome and region, inherited with minimal chance of recombination (Stram et al. 2017 ; Garg et al. 2021 ). Further, by assessing and validating phenotypic effects of the haplotypes through haplo-pheno analysis, favourable haplotypes has been identified (Abbai et al. 2019 ; Sinha et al. 2020 ). The interaction between nitrogen and phosphorus significantly influences plant growth and development (Krouk and Kiba, 2020 ). Although several genomic regions associated with either N or P deficiency have been identified, studies on their combined effects in rice remain limited. Our previous research identified key N- and P-related genes through marker-trait associations under simultaneous N and P deficiency conditions (Chidambaranathan et al. 2024 ). Given that OsSPX-MFS3 is well-characterized for its role in P deficiency and recent findings suggest that SPX proteins may regulate nitrate-dependent Pi homeostasis, we hypothesize that OsSPX-MFS3 may also be involved in seedling responses under combined N and P deficiency conditions. Therefore, this study aims to identify genetic variants and favorable haplotypes of the OsSPX-MFS3 gene ( Chr06: 1552973–1560896 ) associated with seedling traits under low-N and low-P conditions. Our findings provide insights into the coordinated utilization of nitrogen and phosphorus in rice, emphasizing the significance of nutrient signaling networks in improving nutrient use efficiency. Materials and methods Experimental design In this research study, 223 indica rice accessions ( ind1 A : 20 accessions , ind1B: 20 accessions, ind2: 37 accessions, ind3: 74 accessions, and indx: 72 accessions) were obtained from the 3 K SNP Seek Database (Mansueto et al. 2017 ). The phenotypic evaluation was conducted in the low N and P red soil tank at the ICAR-National Rice Research Institute, Odisha (20.4537° N, 85.9338° E) as reported previously (Chidambaranathan et al. 2024 ). Six seeds per hill and three hills per accession were sown in direct-seeded rice (DSR) method with a spacing of 15 cm × 20 cm plots in an area of 30 m 2 . The seeds taken for sowing were uniformly dried in a hot air oven for 5 days at 45 degrees Celsius and taken for the sowing. The experiment was conducted in augmented randomized block design in four blocks with three checks (ENT-621, ENT- 396 and ENT-17) (Chidambaranathan et al 2024 ), the checks were replicated 3 times in each block. The accessions were line sown with three hills per accession. This study was carried out in two different seasons, i.e. kharif (2023) and rabi (2023-24) seasons to the response of the indica rice accessions under low nitrogen and low phosphorus conditions. The study area plot received recommended doses of potassium at a rate of 40 kg ha −1 K 2 O prior to sowing and there was no dosage of nitrogen and phosphorus. Further, normal management practices with an irrigation schedule for every three days once or as required and other management practices were followed as recommended. The seedling growth was observed in 28 and 42 days after sowing (DAS) for eight different phenotypic traits. Soil properties Soil samples were collected from the Ap horizon (0–15 cm depth) before sowing of the crop. Six soil (~500 g of soil) samples were collected randomly from the experimental low N and low P soil tank. These soil samples were air dried in shade condition and passed through a 2-mm sieve. Soil samples were analysed using standard procedures. By using a soil-water suspension (1: 2) (Horney et al. 2005 ), the pH of the soil and EC were determined. Soil organic carbon was determined by following the procedures of Walkley and Black ( 1934 ) (Walkley and Black 1934 ). Available soil nitrogen in soil was determined by following the procedures of Subbiah and Asija ( 1956 ) (Subbiah and Asija 1956 ). Available P was determined by procedures of Bray and Kurtz ( 1945 ) (Bray and Kurtz 1945 ), and available soil potassium was determined by using neutral normal ammonium acetate extractable potassium (K) (Hanway 1952 ). The properties of the soil are summarized in Table 1 . Table 1. Summary of initial soil properties under low N and low P condition Sl. No Soil property 1 pH 6.14 2 EC (mS m −1 ) 238 3 SOC (%) 0.41 4 Available soil N (kg ha −1 ) 158.89 5 Available soil P (kg ha −1 ) 12.66 6 Available soil k (kg ha −1 ) 135.26 Open in a new tab pH Potential of hydrogen, EC electrical conductivity, SOC soil organic carbon, N Nitrogen, P Phosphorus, K Potassium Phenotypic traits The phenotypic measurements which included shoot length (SL), root length (RL), shoot area (SA), root area (RA), shoot dry weight (SDW), root dry weight (RDW), SPAD (chlorophyll index), and number of leaves (NL) were measured. Plant samples were collected at 28 and 42 days after sowing (DAS) in 3 replications of each accession. The shoot length was measured using a 1-m long scale. Plant samples were scanned using a Canon LiDE 400 scanner and then RL, SA, RA were measured using ImageJ software. SDW and RDW were measured from an oven-dried (at about 45 0 C) samples separately for the shoots and roots. Samples were completely oven-dried, weighed, and the results were expressed in milligrams (mg). The number of leaves per plant was counted manually. The SPAD readings were measured by a chlorophyll meter (SPAD-502plus, Minolta Co., Japan) (Dadhich et al. 2023 ). Haplotype analysis The candidate gene is OsSPX-MFS3 ( SPX-MFS protein3, SPX-Major Facility Superfamily3) located chr06:1554861–1561163 (+strand) with locus id LOC_Os06 g0129400 (Os06 t0129400-01 and Os06 t0129400-02). A haplotype analysis was conducted on the OsSPX-MFS3 gene region to group the accessions into different haplotypes. The SNP data for the gene were retrieved from the 3 K_RG panel of the Rice SNP Seek Database ( https://snp-seek.irri.org/_snp.zul ). Haplotype groups or haplotypes were identified from imputed SNP variations of 223 rice accessions and only those haplotypes with more than five accessions were considered in the analysis (Li et al. 2023 ). Haplotypes between the significantly associated loci in the genes were manually identified and analysed for their mean significant difference using the ANOVA p < 0.05 levels of significance. Genetic diversity and Haplotype Network analysis The retrieved SNP data of 223 indica rice accessions of gene OsSPX-MFS3 (LOC_Os06 g0129400) from SNP-seek database was utilized for the genetic diversity and haplotype analysis. There are 27 SNPs in the promoter (2k bp upstream regions), 7 SNPs in the UTR regions, 13 SNPs are in the coding region, and 52 SNPS are in the intron region of the SPX-MFS3 . The SNP file was converted into the FASTA file format and were aligned using the ClustalW multiple pairwise alignment function with gap penalty 15 and gap extension 6.66 in MEGA11 software (Tamura et al. 2021 ). The aligned file was exported in mega file format for genetic diversity study and haplotypic grouping in DnaSP software (DNA Sequence Polymorphism) (Rozas et al. 2017 ). Haplotype diversity (Hd), nucleotide diversity (π), Tajima’s D, FLD, FLF values were calculated using DnaSP6(Zhang et al. 2024 ). The resulting list of haplotype groups was used for haplotype network construction using “Pegas” R package (Paradis, 2010). Statistical analysis Descriptive statistics were conducted using the data analysis tool in MS Excel. Single-factor analysis of variance (ANOVA) was used to understand the significant interactions between the traits and accessions. Principal component analysis (PCA) was performed using the ‘prcomp’ function (Quicke et al. 2020 ) and visualized through the ‘ggfortify’ package in R (Tang et al. 2016 ). Box plot was constructed using the Microsoft Excellstudy the significance between haplotypes and traits. The mean difference between the haplotypes was statistically analysed through analysis of variance (ANOVA) p -value <0.05 level of significance. Tukey HSD test was performed between haplotype by using the Xlstat software. Results Genetic diversity and haplotypic network analysis of SPX-MFS3 gene The nucleotide diversity (π) of the OsSPX-MFS3 gene was analysed to evaluate the degree of polymorphism in the 223 accessions using 99 SNPs. It showed 47 segregating SNPs (polymorphic SNPs) and the nucleotide diversity(π) of the gene was 0.3595. Further, promoter, UTR, exon, and intron nucleotide diversity identified was 0.122, 0.115, 0.135, and 0.095, respectively. To gain further insights into the presence of purifying or balanced selection in the SPX-MFS3 gene within the population, Tajima’s D value for the OsSPX-MFS3 gene was negative and significant (− 1.82982*). However, Tajima’s D value for the promoter (− 0.65), UTR (− 0.75), exon (− 1.27), and intron (− 1.61) were though negative but not significant. Besides, analysis of Fu and Li’s D and F test showed a significant and highly negative value (FLD: − 3.12* and FLF: − 2.98**) only for the exon region of the gene (Table 2 ) which indicates an excess of rare variants. Further, haplotypes analysis identified eight out of 20 as major haplotypes with haplotype diversity (Hd) of 0.095. A closely connected network was observed among the haplotypes of indica rice accessions (Fig. 1 ). Table 2. Summary of genetic diversity (Tajima’s, Nucleotide Diversity, Fu and Li’D and Fu and Li’s Ftest). (Statistically significance has been marked i.e. *, p <0.05 and **, p <0.02) No. of SNPs Tajima’s Nucleotide diversity Fu and Li’s D Fu and Li’s F SPX-MFS3 99 − 1.8298* 0.13595 − 0.58692 − 1.22982 Exon 13 − 1.27088 0.13593 − 3.1215* − 2.98937** Intron 52 − 1.61405 0.09575 1.05608 0.15971 UTR 7 − 0.7556 0.15588 0.22537 − 0.18768 Promotor 27 − 0.65291 0.12201 1.07853 0.50851 Open in a new tab Fig. 1. Open in a new tab Haplotype network analysis of the OsSPX-MFS3 gene was conducted using 223 Indica rice accessions, revealing the presence of eight major haplotypes. Vertical lines indicate the number of SNPs differing between the two haplotypes. Hap – Haplotype. Different haplotypes are presented as different colours Analysis of variance, correlation, and mean difference analysis between the haplotypes of OsSPX-MFS3 Correlation analysis for the eight major haplotypes under low N and low P conditions showed SDW was correlated with RDW (r=0.89** in kharif and r=0.94*** in rabi seasons), SL was highly correlated with SA (r=0.93***), SA with both SDW (r=0.69*) and RDW (0.74*) in kharif 28DAS. In rabi 28DAS, SL was found to be highly correlated with RDW (r=0.94***) and SDW (r=0.75*). Further, SA was highly correlated with three traits namely RA (r=0.86**), SDW (r=0.90**) and RDW (r=0.91**) and also RA was correlated with two traits namely SDW(r=0.76*) and RDW (0.90**). In 42DAS, root length (RL) was found to be highly correlated with three traits namely SA (Kharif: 0.71*; rabi:0.78*), RA (Kharif:0.75*; rabi:0.87**), and RDW (Kharif:0.71*; rabi:0.91**). Similarly, RDW was highly correlated with four traits namely RL (kharif:0.71*; rabi:0.91**), SA (kharif:0.88**; rabi:0.82*), RA (kharif:0.77*; rabi:0.91**), and SDW (kharif:0.85**; rabi:0.94***) (Fig. 2 ). The eight major haplotypes were analysed for eight phenotypic traits (Shoot length (SL), root length (RL), shoot area (SA), root area (RA), shoot dry weight (SDW), root dry weight (RDW), SPAD and number of leaves (NL)) under a combination of low N and low P conditions in 28 and 42 DAS in both kharif and rabi seasons (Tables 3 and 4 ). The haplotype, Hap_1 consisted of 10 accessions, Hap_2 and Hap_8 each had 34 accessions, Hap_3 and Hap_5 each had 8 accessions, Hap_4 and Hap_7 consisted of 9 and 15 accessions, respectively. Further, Hap_6 is the major haplotype with 73 indica rice accessions (Fig. 3 ). Besides, within the coding region of OsSPX-MSF3 , we identified seven functional (non-synonymous) SNPs (referred to as fSNPs hereafter). One of these fSNPs (G/T variation at the position 1556763 in exon 2) was present in Hap_4. Similarly, another haplotype, Hap_2 was characterized by 4 SNPs in the non-coding regions of the gene (C/T, A/T, T/C, and C/T at positions 1555037, 1555863, 1555935, and 1557941, respectively, and a fSNPs (G/T substation at position 1557453 in exon 3). Additionally, Hap_8 had an fSNP (T/G at position 1558112 and A/T coding in position 1560628 in exons 5 and 10, respectively) which is found in 34 accessions. Similar to Hap_2, Hap_4 contains 7 SNPs in non-coding regions and a fSNP (C/T coding position of 1560074 in exon 9) present in 9 accessions. Most superior Hap_5 had an fSNPs A/A at coding position 1558436 in exon present 8 accessions. The most prevalent haplotypes, Hap_6 and Hap_7, have fSNPs (C/T coding for exon 10 in position 1560550) and consist of 73 and 15 accessions, respectively. Further, Hap_7 has one SNPs in non-coding regions C/G at position 1560550 (Fig. 3 ) (Tables 5 and 6 ). Fig. 2. Open in a new tab Pearson correlation coefficients to assess the relationships among various phenotypic traits across eight haplotypes under low nitrogen (N) and phosphorus (P) conditions (i) 28 DAS Kharif (ii) 28 DAS Rabi (iii) 42 DAS Kharif and (iv) 42 DAS Rabi. (SL-Shoot Length, RL-Root Length, SA-Shoot Area, RA-Root Area, SDW-Shoot Dry Weight, RDW-Root Dry Weight, SPAD-Chlorophyll index, NL-No. of leaves). *, **, *** indicates level of significance at 5%, 1%, 0.01%, respectively. Black lines represent regression curve and red line represents distribution Table 3. Mean data of each phenotypic character considered in this experiment for 8 haplotypes under low N and low P condition in 28DAS Kharif and Rabi Haplotype 28DAS_Kharif 28DAS_Rabi SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL Hap_1 29.22 8.16 21.08 4.05 85.41 30.04 30.09 4.40 29.88 10.64 11.24 3.71 111.97 35.24 24.68 4.30 Hap_2 26.30 7.38 17.47 3.91 75.69 25.98 28.89 3.99 30.25 12.38 14.38 5.26 141.33 61.42 22.47 4.58 Hap_3 27.24 8.66 16.87 3.80 63.97 15.62 28.47 3.83 32.15 12.58 19.56 4.73 215.06 79.01 25.66 4.42 Hap_4 29.53 6.47 20.94 3.86 78.70 30.57 28.61 3.74 28.26 11.87 16.51 4.51 126.54 47.62 21.70 4.44 Hap_5 26.88 8.18 18.60 5.93 77.16 26.93 27.86 4.21 33.20 13.22 25.52 7.84 233.36 106.10 26.73 4.50 Hap_6 27.80 7.31 18.92 4.33 80.13 28.23 28.35 4.10 30.19 12.85 13.31 4.63 148.52 64.03 26.58 4.43 Hap_7 25.65 8.10 15.53 3.83 63.02 21.84 28.70 3.87 32.41 11.30 15.06 4.65 127.93 59.08 24.58 4.42 Hap_8 28.10 6.46 18.88 3.59 71.30 21.30 27.95 4.10 30.84 12.32 13.91 3.48 131.87 48.18 24.47 4.44 Mean 27.59 7.59 18.54 4.16 74.42 25.06 28.62 4.03 30.90 12.15 16.19 4.85 154.57 62.59 24.61 4.44 ANOVA P value 0.53 0.37 0.66 0.40 0.66 0.20 0.73 0.19 0.65 0.20 0.0034 0.026 0.002 0.012 0.0054 0.59 Significance level ns ns ns ns ns ns ns ns ns ns ** * ** * ** ns Open in a new tab One-way ANOVA P value statistically significant asterisks has been marked i.e., ns for non-significant ( P > 0.05), * for P ≤ 0.05, and ** for P ≤ 0.01 SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves Table 4. Mean data of each phenotypic character considered in this experiment for 8 haplotypes under low N and low P condition in 42DAS Kharif and Rabi Haplotype 42DAS_Kharif 42DAS_Rabi SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL Hap_1 49.00 8.07 40.72 5.66 229.77 74.52 29.21 4.47 40.89 10.93 19.69 6.33 266.90 74.21 33.70 4.87 Hap_2 43.25 9.99 50.08 7.73 290.71 102.24 25.69 4.86 42.40 13.21 18.53 7.10 273.34 104.80 29.50 5.38 Hap_3 45.58 8.78 26.04 4.87 154.21 57.15 26.47 4.21 42.90 11.41 20.40 5.65 266.65 90.06 35.18 5.33 Hap_4 45.04 10.42 52.02 11.71 282.55 124.95 28.61 4.96 42.05 13.45 20.01 6.21 255.23 97.00 28.57 5.48 Hap_5 36.04 10.07 42.68 9.03 199.43 91.88 28.32 4.83 41.68 17.63 33.74 11.03 404.00 179.55 33.43 5.92 Hap_6 44.95 9.47 50.32 7.87 273.96 114.74 26.81 5.02 43.66 13.66 23.61 8.67 322.39 150.71 33.78 5.73 Hap_7 41.10 9.47 37.15 7.40 256.71 117.26 27.33 5.33 43.58 11.60 24.66 5.58 263.28 95.29 33.73 5.11 Hap_8 46.90 10.72 58.30 7.86 299.91 113.57 25.35 4.99 46.41 12.59 23.97 8.53 295.40 110.81 35.49 5.50 Mean 43.98 9.62 44.66 7.77 248.41 99.54 27.22 4.83 42.95 13.06 23.08 7.39 293.40 112.80 32.92 5.42 ANOVA P value 0.014 0.37 0.05 0.029 0.44 0.52 0.17 0.15 0.51 0.031 0.17 0.20 0.69 0.23 0.034 0.61 Significance level * ns * * ns ns ns ns * ns ns ns ns * ns Open in a new tab One-way ANOVA P value statistically significant asterisks has been marked i.e., ns for non-significant ( P > 0.05), * for P ≤ 0.05, and ** for P ≤ 0.01 SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves Fig. 3. Open in a new tab Gene structure and haplotypes of OsSPX-MFS3 ( Os06 go129400, Chr: 06) under combination of low N and low P conditions. SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index. Yellow rectangle - promoter, grey box - UTRs, blue box - exon, and black horizontal line - introns. Numbers in chromosome position represents SNP ids, reference nucleotides are highlighted in green fill color column, and amino acid (AA) variations are represented in coloum without fill color. Green-filled boxes represent haplotypes with the highest mean value for respective traits Table 5. Summary statistics of the phenotypic characters considered for this experiment for 8 haplotypes under low N and low P condition in 28DAS Kharif and Rabi 28DAS_Kharif 28DAS_Rabi SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL Mean 27.59 7.59 18.54 4.16 74.42 25.06 28.61 4.03 30.90 12.14 16.19 4.85 154.57 62.58 24.61 4.44 SD 1.35 0.82 1.91 0.75 7.83 5.12 0.69 0.22 1.61 0.84 4.49 1.34 44.57 21.90 1.80 0.08 Minimum 25.65 6.46 15.53 3.59 63.02 15.62 27.86 3.74 28.26 10.64 11.24 3.48 111.97 35.24 21.70 4.30 Maximum 29.53 8.66 21.08 5.93 85.41 30.57 30.09 4.40 33.20 13.22 25.52 7.84 233.36 106.10 26.73 4.58 CV (%) 4.90 10.83 10.30 17.92 10.53 20.41 2.42 5.37 5.20 6.95 27.73 27.58 28.83 35.00 7.30 1.77 Open in a new tab SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves, CV Coefficient variance Table 6. Summary statistics of the phenotypic characters considered for this experiment for 8 haplotypes under low N and low P condition in 42DAS Kharif and Rabi 42DAS_Kharif 42DAS_Rabi SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL SL (cm) RL (cm) SA (cm 2 ) RA (cm 2 ) SDW (mg) RDW (mg) SPAD NL Mean 43.98 9.62 44.66 7.76 248.41 99.54 27.22 4.83 42.94 13.06 23.08 7.39 293.40 112.80 32.92 5.41 SD 3.97 0.87 10.17 2.07 50.64 23.53 1.40 0.35 1.68 2.11 4.87 1.89 49.66 34.90 2.52 0.33 Minimum 36.04 8.07 26.04 4.87 154.21 57.15 25.35 4.21 40.89 10.93 18.53 5.58 255.23 74.21 28.57 4.87 Maximum 49.00 10.72 58.30 11.71 299.91 124.95 29.21 5.33 46.41 17.63 33.74 11.03 404.00 179.55 35.49 5.92 CV (%) 9.02 9.06 22.77 26.72 20.38 23.64 5.14 7.21 3.92 16.12 21.11 25.60 16.938 30.93 7.67 6.09 Open in a new tab SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves, CV Coefficient variance In the promotor region observed that Hap_1contains three non-coding regions having G/A,C/G and T/A at position 1553011, 1553480 and 1554811, respectively. However, Hap_2 had 2 SNPs noncoding region at G/A and T/C at position 1553009 and 1553613, respectively. Hap_6 and Hap_7 one SNPs T/A at position 1554811. Hap_3 having 2 noncoding (C/T and T/A at the position 1554688 and 1554817, respectively) and Hap_4 having one SNPs non-coding region (T/C at the position 1553613). Thus, Hap_5 having 12 SNPs noncoding region having effect of promotor regions that result phenotypical changes of accessions (Fig. 3 ). Mean difference analysis between the major haplotypes under combination of low N and P conditions showed Hap_5 has the largest mean value for the traits namely root length (17.6 cm), shoot area (25.5 cm2), root area (7.8 cm2), shoot dry weight (233.4 mg), root dry weight (106.1 mg) and SPAD (26.7). Further, Hap_1 (49.0 cm) and Hap_8 (46.89 cm) mean values were higher for shoot length at kharif 42DAS, Hap_4 mean value were higher for root area (11.70 cm2) at kharif 42 DAS. The mean trait values of the haplotypes were compared using a one-way ANOVA test ( p <0.05), which revealed a significant difference between the haplotypes and traits (Fig. 4 ). The analysis of variance between the haplotypes showed none of the haplotypes showed statistical significance for eight traits in the kharif season at 28 DAS. However, in rabi season for 28 DAS, shoot area (0.003**), root area (0.02*), shoot dry weight (0.002**), root dry weight (0.012*), and SPAD (0.005**) showed statistical difference between the haplotypes. Similarly, shoot length (0.014*), shoot area (0.05*) and root area (0.029*) showed a significant difference between the haplotypes in kharif seasons at 42 DAS. In contrast, only two traits namely root length (0.031*) and SPAD (0.034*) showed statistical significance between the haplotypes in rabi at 42 DAS. Fig. 4. Open in a new tab Phenotypic effects of OsSPX-MFS3 haplotypes under low N and P conditions. Box plot represents the differences between the haplotypes on (i) Shoot Area (ii) Root Area (iii) Shoot Dry Weight (iv) Root Dry Weight and (v) SPAD in 28DAS of Rabi. (vi) Shoot Length (vii) Shoot Area and (viii) Root Area in 42DAS Kharif. (ix) Root Length and (x) SPAD in 42DAS Rabi. The significant differences between haplotypes were tested using one-way ANOVA ( p < 0.05). Additionally, the Tukey HSD test was performed for multiple comparisons among haplotypes, with significant differences marked using alphabetic groupings Phenotypic effect of OsSPX-MFS . Marker trait association The total number of nucleotide SNP variations in the OsSPX-MFS3 gene among the 223 accessions was 99 SNPs. These SNPs were used in the marker trait association analysis to identify the significant associations for different traits in both seasons. Seven significant trait associations were found for shoot area, shoot dry weight, root length, root area, root dry weight, SPAD, and number of leaves under low N and low P conditions using MLM and Blink models. Besides, root length (S6_1558112) and number of leaves (S6_1557453) associations was observed in kharif 28 DAS, and five SNPs association was observed for shoot area (S6_1557453, S6_1555935, S6_1557941, S6_1555863, and S6_1555037) in rabi 28 DAS. Further, three association were identified each for shoot dry weight (S6_1555300, S6_1557942, and S6_1560550), root dry weight (S6_1560550, S6_1555300 and S6_1557942) and one for root area (S6_1555300) in kharif 42 DAS. Additionally, two associations were observed for SPAD (S6_1558410 and S6_1560592) with phenotypic variance 10.7% and 48.7%, respectively (Table 7 ). Table 7. Marker and trait associations for low N and Low P conditions SL. No. Trait Marker Chromosome Position p . value effect Observation MarkerR 2 Season Model 1 RL S6_1558112 6 1558112 0.02352 − 0.771 223 3.411 Kharif 28 DAS MLM 2 NL S6_1557453 6 1557453 0.04788 − 2.697 223 2.697 Kharif 28 DAS MLM 3 SA S6_1557453 6 1557453 0.00441 − 2.683 223 5.020 Rabi 28 DAS MLM 4 SA S6_1555935 6 1555935 0.00446 2.395 222 5.011 Rabi 28 DAS MLM 5 SA S6_1557941 6 1557941 0.00446 − 2.395 222 5.011 Rabi 28 DAS MLM 6 SA S6_1555863 6 1555863 0.00456 − 18.222 222 4.992 Rabi 28 DAS MLM 7 SA S6_1555037 6 1555037 0.00522 23.830 223 4.862 Rabi 28 DAS MLM 8 SPAD S6_1558410 6 1558410 0.00034 − 9.659 223 10.713 Rabi 28 DAS Blink 9 SPAD S6_1560592 6 1560592 0.00034 − 9.659 223 48.784 Rabi 28 DAS Blink 10 SDW S6_1555300 6 1555300 0.00274 − 56.800 223 5.504 kharif 42 DAS MLM 11 SDW S6_1557942 6 1557942 0.0075 − 32.600 223 4.545 kharif 42 DAS MLM 12 RDW S6_1560550 6 1560550 0.00778 − 15.600 223 4.478 kharif 42 DAS MLM 13 SDW S6_1560550 6 1560550 0.01209 50.240 223 4.092 kharif 42 DAS MLM 14 RDW S6_1555300 6 1555300 0.01745 − 12.100 223 3.719 kharif 42 DAS MLM 15 RA S6_1555300 6 1555300 0.02317 1.056 223 3.369 kharif 42 DAS MLM 16 RDW S6_1557942 6 1557942 0.02987 3.686 223 3.217 kharif 42 DAS MLM 17 NL S6_1555676 6 1555676 3.66E-12 − 0.952 223 7.186 Kharif 42 DAS Blink Open in a new tab RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves, DAS days after sowing PCA analysis The OsSPX-MFS3 gene, represented by eight haplotypes, was evaluated using principal components analysis (PCA) to assess trait grouping during the kharif and rabi seasons at 28 and 42 days after sowing (DAS). Under low nitrogen (N) and low phosphorus (P) conditions at 28 DAS in the kharif season, the first three principal components collectively accounted for 87.4% of the total variation among the haplotypes (Fig. 5 (i)). In particular, the first (PC1) and second (PC2) components individually explained approximately 48.7 and 22.3% of the variation, respectively (Supplementary Table 2a). Bi-plot analysis for kharif 28 DAS showed that shoot length, shoot area, shoot dry weight, and root dry weight were highly correlated in principal component 1 (PC1)). Besides, within PC1, root area and SPAD values exhibited a weaker correlation. Further, root length in PC2 was largely negatively correlated with shoot length in PC1 (Fig. 5 (i); Supplementary Table 4a). Additionally, the superior haplotypes Hap5 was grouped with the root area in PC1 for the kharif 28 DAS. Fig. 5. Open in a new tab Principal component analysis (PCA) illustrates the variance among eight haplotypes based on eight phenotypic traits under low nitrogen (N) and phosphorus (P) conditions in (i) 28DAS kharif, (ii) 28DAS rabi, (iii) 42DAS kharif and (iv) 42DAS rabi. SL Shoot length, RL Root length, SA Shoot area, RA Root area, SDW Shoot dry weight, RDW Root dry weight, SPAD Chlorophyll index, NL No. of leaves. PC1 Principal component 1, PC2 Principal component 2, Hap Haplotypes During the rabi season at 28 DAS, the first two principal components explained a cumulative 91.6% of the variation (Supplementary Table 2b). Notably, the number of leaves, root length, root area, shoot area, and root dry weight were strongly intercorrelated and clustered within a single component (PC1). Moreover, shoot dry weight, shoot length, and SPAD were also grouped in PC1. Similar to kharif 28DAS, shoot and root length were weakly positively correlated in PC1. Besides, Hap5 was found to be present along with shoot and root dry weight in PC1. In 42DAS kharif conditions, the first three principal components contributed to 89.6 percent variation between the haplotypes (Fig. 5 (iii), Supplementary table 2c). The traits namely shoot and root length were negatively correlated and Hap5 was grouped along with SPAD values(Fig. 5 (iii)). In 42DAS rabi conditions, the first three principal components contributed to 95.3 percent variations (Fig. 5 (iv) and Supplementary table 2 d). The traits such as SL, SPAD, SA, RA, and SDW were grouped together in the first principal component (PC1) and RL, RDW, and NL were grouped and highly correlated in the second principal component (PC2) (Fig. 5 (iv)). Moreover, shoot and root length was negatively correlated and Hap5 was associated with root length and no. of leaves. In all four season PCA analysis, Hap5 was grouped in one component and most of the remaining haplotypes were grouped in different components. Interestingly, two traits namely shoot and root dry weight were highly correlated in all two seasons and time points indicative of a major associated trait for combination of low N and low P tolerance. Additionally, Hap5 with japonica type promoter was found to be highly correlated with root dry weight, shoot dry weight, and root length. Similarly, Hap2 and Hap4 with non-synonymous substitution, was found to be grouped together in rabi 28 DAS, kharif 42 DAS, and rabi 42 DAS (Fig. 5 ). OsSPX-MFS3 protein structure and promoter analysis The OsSPX-MFS3 gene, containing 99 SNPs across its promoter, UTR, exon, and intron regions, consists of two non-synonymous SNPs (Chr06:1556763 and Chr06:1557453). These SNPs resulted in amino acid changes leucine to phenylalanine at position 25 (Leu25Phe in HAP4) and alanine to serine at position 97 (Arg97Ser in HAP2). The remaining haplotypes showed no differences in the protein sequence. The secondary structure of the reference OsSPX-MFS3 protein and its mutants was predicted using PSIPRED tool. The wild-type (reference) protein and Leu25Phe mutant retains two β-strands, and the Arg97Ser mutant has only one β-strands. The α-helices showed almost similar structure with only slight variations across all three protein models. Further, tertiary structure predictions( https://swissmodel.expasy.org/ ) reveal minor structural deviations between the reference and mutant proteins (Supplementary table 4). The QMEANDisCo scoring function was employed to assess structural quality, providing both global and local quality estimates. The reference protein scored 0.50 ± 0.05, while the Leu25Phe and Arg97Ser mutants scored 0.49 ± 0.05 and 0.47 ± 0.05, respectively, indicating minor reductions in structural reliability in the mutant proteins. The 2kb promoter analysis of OsSPX-MFS3 haplotypes showed distinct cis-regulatory elements specifically in HAP5 as compared to other haplotypes (Supplementary Table 5). The HAP5 promoter cis-regulatory elements are similar to japonica type promoter and the remaining haplotypes were similar to indica type promoter. The japonica type promoter of OsSPX-MFS3 (HAP5) comprised of cis-regulatory elements such as ERE (Estrogen Responsive Element), MYB (cell differentiation, development, and stress responses), ABRE(cis-acting element involved in the abscisic acid responsiveness), CAAT-box(protein that binds to the CAAT-box DNA site to regulate gene transcription), MYC(MYC transcription factor, a key regulator of gene expression involved in numerous cellular processes) and O2-site(cis-acting regulatory element involved in zein metabolism regulation). In contrast, remaining indica type promoter haplotypes consist of G-Box (cis-acting regulatory element involved in light responsiveness), as-1(response to environmental stresses and signalling molecules like salicylic acid and auxins), CGTCA-motif and TGACG-motifs (Fig. 7 ). This indicates the expression variation between indica and japonica type promoters could play an important role in trait responses. Fig. 7. Open in a new tab Predicted mutation effect and their phenotypic impact of OsSPX-MSF3 gene under low N and P condition at the seedling stage. A Non-synonymous SNPs positions (1556763 and 1557453) of the gene. Yellow rectangle - promoter, grey box - UTRs, blue box - exon, and black horizontal line - introns. Pink fill color represents alpha helix secondary structure; yellow fill color represents β-turns. Protein tertiary structure is given beside the secondary structure. Dark blue color in tertiary structure represents good superposition, green, yellow, and red represents alignment divergence with the reference structure. WT-Wild type, HAP-Haplotypes b Schematic promoter representation of cis-regulatory motifs of OsSPX-MFS3 , different cis-regulatory motifs are represented by different symbols. Alignment of the promoter SNPs are given below the schematic promoter representation c representative seedlings of superior HAP5 accessions showing tolerance under the combination of low P and N conditions d representative seedlings of remaining inferior haplotypes Discussion Several previous reports of OsSPX-MFS3 highlighted its physiological function as involved in intracellular P homeostasis and vacuolar Pi trafficking in rice (Wang et al. 2015 ; Guo et al. 2023b ). However, most of these studies evaluated the OsSPX-MFS3 function under normal and low P conditions. This study identified OsSPX-MFS3 haplotypes differed significantly for various traits under the combination of N and P deficiency at the seedling stage in rice. This indicates haplotype genetic differences could be associated with the regulation of seedling growth under low N and P conditions. Besides, the negative Tajima’s D value also indicates the presence of rare genetic variants and haplotypes which could be related to differential responses of the OsSPX-MFS3 . In support of these findings, negative Tajima’s D values were also observed for nitrate reductase (NR2) gene in rice (Gao et al. 2019 ), phosphorus-Starvation Tolerance 1 gene ( PSTOL1 ) from Oryza rufipogon (Neelam et al. 2017 ), high-affinity ammonium transporter gene (OsAMT1;1) in rice (Ding et al. 2011 ). In maize, the combination of low N and low P attenuates the characteristic and highly conserved phosphorus starvation response (PSR) observed in low P conditions (Torres-Rodríguez et al. 2021 ). In rice, PSR is characterized by enhanced root growth and reduction in shoot growth and biomass (Mukkawar et al. 2024 ). This attenuation of PSR assists in preventing the hyperaccumulation of P under low N conditions as reported in maize (Du et al. 2018 ). Interestingly, one of the haplotypes ( OsSPX-MFS3 : Hap5) mirrored the PSR response even in the combination of low N and low P conditions characterized by reduced shoot length and relatively higher root length. This response ( OsSPX-MFS3 : Hap5) is in contrast to the findings of Torres-Rodríguez et al. 2021 , observation in maize wherein under both N and P deficiency, low N response was highly pronounced. Therefore, OsSPX-MFS3 :Hap5 showing PSR response might be associated with enhanced sequestration of P in vacuoles relative to other haplotypes. On the other hand, Hap5 might be insensitive to low N conditions and highly pronounced PSR. Interestingly, rather than the protein sequence variation between the haplotypes, HAP5 promoter was found to be similar to the japonica promoter type and might be associated with differential expression pattern relative to indica type promoters. In support of this observation, previous studies have identified promoter differences between indica and japonica accessions of genes namely OsDNR1 , OsMYB61 , OsARE1 , OsAAP3 , OsAAP5 , and OsTCP19 associated with nitrogen use efficiency (Hu et al, 2023 ). Therefore, promoter characterization of Hap5 might provide better insights into the combined low N and low P response in rice. The rice mutant lines of OsSPX-MFS3 of Nipponbare showed reduced biomass and plant height under low P conditions (Guo et al. 2023a ). Though this study also observed reduced shoot length in Hap5, biomass (RDW=106.1 mg) was relatively higher indicating the differential effects of Hap5 in regulating the seedling response in low N and low P conditions. Similar findings of root biomass variations between the haplotypes have been reported for OsPht1.2 and OsEXPA12 genes but only under low N conditions in rice (Anandan et al. 2022 ). The haplotype significance analysis showed shoot and root length, shoot and root dry weight, and chlorophyll index (SPAD) significantly differed between the OsSPX-MFS3 haplotypes. In support of these findings, our previous study also reported many significant marker trait associations for shoot and root-related traits under low N and low P conditions (Chidambaranathan et al. 2024 ). This indicates the confounding effects of OsSPX-MFS3 in regulating the seedling growth under low N and low P conditions. However, most of the significant differences between the haplotypes were observed only in the rabi rather than the kharif season. In one of the previous reports, yield and other yield-attributing traits were found to be relatively higher in the dry season compared to the wet season in rice for the same management practices (Calayugan et al. 2024 ). Similar findings of dry and wet seasonal differences in yield for different levels of N was also observed in rice (Mboyerwa et al. 2022 ). Similar observations of seasonal effects on the N and P uptake in sunflower were correlated with growing degree days and NPK uptake only in the spring season (dry season) and negatively correlated during the autumn season (Hassan and Kaleem 2014 ). These reports support our findings of significant differences between the OsSPX-MFS3 haplotypes in the kharif (wet season) and rabi season (dry season). Besides, it provides clues about the differential response of OsSPX-MF3 under low N and low P conditions might well also be regulated by growing degree days and sunshine hours. This observation requires in-depth investigation. Root dry weight showed significant correlations with shoot dry weight, shoot and root area under low N and low P conditions at 28 and 42 days after sowing. This indicates root dry weight would be used as a major selection trait for the identification of the tolerant accessions under both N and P deficient conditions and three tolerant accessions (IRGC 131940, IRGC126966, and IRGC 122165) for enhanced shoot and root growth under deficiency conditions in both the kharif and rabi seasons (Fig. 6 ). This finding not only supported our previous findings (Chidambaranathan et al. 2024 ), but peculiarly relative to nitrogen deficiency, upland rice variety (Zhu et al. 2024 ), qSOR-NILs (Oo et al. 2024 ), and low P tolerant rice varieties (Deng et al. 2020 ) showed higher root dry weight under low P conditions. This indicates low P characteristic response of higher root dry weight is exhibited by indica rice accessions even under low N and low P conditions. This finding was supported by recent findings that the belowground biomass increase is a major adaptation strategy under nutrient deficiency conditions in tomato which assists not only for nutrient foraging but also for carbon budgeting and promotion of mycorrhizal interaction (Wang et al. 2023 ). Therefore, high root biomass of OsSPX-MF3 Hap5 might be a better adaptation strategy under low N and P adaptation conditions relative to other haplotypes. Fig. 6. Open in a new tab Tolerant and susceptible indica rice accessions identified at the seedling stage under low nitrogen (N) and phosphorus (P) conditions. a Tolerant accessions – accessions exhibiting robust shoot and root growth under nutrient-deficient conditions. b Susceptible accessions – accessions displaying restricted shoot and root development under low N and P availability. IRGC-accession id, Hap_haplotypes Conclusion In conclusion, shoot and root dry weight was identified as the major trait associated with combined N and P deficiency tolerance response in indica accessions of rice. Further, seasonal effects of the OsSPX-MFS3 haplotypes were also identified indicating environmental effects in the plant’s nutrient deficiency response. Besides, mechanistically one of the superior haplotypes ( OsSPX-MFS3 -Hap5) exhibited phosphorus starvation response even in the low N and low P response in rice. This favourable haplotype also consists of japonica type promoter and could be utilized in rice breeding programs for enhancing the rice seedlings adaptation to N and P deficiency. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 676 KB) (676.1KB, docx) Acknowledgements Authors are thankful to the Director, ICAR-NRRI, Cuttack, Odisha, India for facilitating the requirements of this study. The technical support provided by technical staff and others project staffs of the Crop Improvement Division, ICAR-NRRI is also acknowledged. Abbreviations SL Shoot length RL Root length SA Shoot area RA Root area SDW Shoot dry weight RDW Root dry weight NL Number of leaves DSR Direct seeded rice DAS Days after sowing PCA Principal components analysis PSR Phosphorus starvation response Author contributions Baishnab Charan Muduli : Writing – original draft, Software, Resources, Methodology, Formal analysis, Data curation. Sabarinathan Selvaraj : Data curation, Formal analysis. Subhadarshani Dhall : Data curation, Formal analysis. Shivraj Sahu : Data curation, Formal analysis. Nibedita Swain : Data curation, Formal analysis. Parameswaran Chidambaranathan : Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization and editing. Jitendriya Meher : Experimental design, data collection, and finalization of data; Chinmay Pradhan : Writing – review & editing, Supervision. Dibyendu Chatterjee : Writing – review & editing. Sangita Mohanty : Writing – review & editing. Sanghamitra Samantaray : Writing – review & editing, Supervision, Investigation, Conceptualization. Funding No specific funding has been received for this work Data and materials availability All data are available upon request to the corresponding author. Declarations Conflict of interest The authors declare no financial or non-financial competing interests References Abbai R, Singh VK, Nachimuthu VV et al (2019) Haplotype analysis of key genes governing grain yield and quality traits across 3K RG panel reveals scope for the development of tailor‐made rice with enhanced genetic gains. 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